Inorganic nitrogen is a vital nutrient for plants. Plants take up and assimilate both nitrate and ammonium with nitrate being the predominant form in most agricultural soils (Crawford N M, Glass A D M, Trends Plant Sci., 3: 389-395 (1998)). Nitrate is taken up by roots then transported into cells via transporters from the NRT1 and NRT2 family of nitrate transporters (Forde B G, Biochim. Biophys. Acta, 1465: 219-235 (2000); Tsay Y F et al., FEBS Lett, 581: 2290-2300 (2007)). Once inside the cell, nitrate is reduced to nitrite by nitrate reductase (NIA) then to ammonium by nitrite reductase (NiR). Ammonium is then assimilated into amino acids.
Nitrate has two functions in plants; it serves as a nutrient and as a signal. Plants undergo many physiological and developmental changes when they are exposed to nitrate. One of the fastest responses is reprogramming of the plant transcriptome. When plants are exposed to nitrate, genes in the nitrate assimilation pathway (NRT, NIA, NiR) are induced within minutes (Wang R et al., Plant Physiol., 132: 556-567 (2003); Scheible W R et al., Plant Physiol., 136: 21013(2003483-2499 (2004); Wang R et al., Plant Physiol., 145: 1735-1745 (2007)). Other genes, which are involved in carbon and energy metabolism that support nitrate assimilation, are also rapidly induced (Stitt M, Curr. Opin. Plant Biol., 2: 178-186 (2006) (1999); Wang R et al., Plant Cell, 12: 1491-1509 (2000); Stitt M et al., J Exp Bot., 53: 959-970 (2002); Wang R et al., Plant Physiol., 132: 556-567 (2003); Scheible W R et al., Plant Physiol., 136: 2483-2499 (2004); Wang R et al., Plant Physiol., 136: 2512-2522 (2004); Fritz C et al., Plant J, 46: 533-548 (2006)). Transcriptome analyses have shown that over 1500 genes are rapidly induced or repressed by nitrate and that the processes of pentose phosphate oxidation, glycolysis, trehalose synthesis, nitrogen and amino acid metabolism are most affected (Wang R et al., Plant Physiol., 132: 556-567 (2003); Scheible W R et al., Plant Physiol., 136: 2483-2499 (2004); Wang R et al., Plant Physiol., 136: 2512-2522 (2004); Gutierrez R A et al., J Exp Bot, 58: 2359-2367 (2007); Wang R et al., Plant Physiol., 145: 1735-1745 (2007)). These rapid transcriptome responses provide the basis for the longer-term responses that direct root growth, development and architecture, root to shoot ratios and germination rates (Forde B G, Annu. Rev. Plant Biol., 53: 203-224 (2002); Alboresi A et al., Plant Cell Environ, 28: 500-512 (2005); Filleur S et al., Biochem Soc Trans, 33: 283-286 (2005); Forde B G, Walch-Liu P, Plant Cell Environ., 32: 682-693 (2009)).
Even though nitrate responses have been reported for plants for over forty years, the first biochemical response being reported in 1957 (Tang P S, Wu H Y, Nature, 179: 1355-1356 (1957)), the regulatory genes that mediate these responses are just now being identified. So far, several potential transcription factors, two kinases and a transceptor have been linked to nitrate regulation (Krouk G et al., Curr Opin Plant Biol., 2010/01/23: 10.1016/j.pbi.2009.1012.1003 (2010)). The ANR1 MADS box transcription factor, which is induced by N deprivation, controls lateral root branching in response to nitrate (Zhang H M, Forde B G, Science, 279: 407-409 (1998); Gan Y et al., Planta, 222: 730-742 (2005)). The NIN-like gene NLP7, encoding a potential bZIP DNA binding protein, was identified through its sequence similarity to the nitrate regulatory gene NIT2 in Chlamydomonas (Camargo A et al., Plant Cell, 19: 3491-3503 (2007)) and functions in nitrate induction of several nitrate assimilatory genes (Castaings L et al., Plant J, 57: 426-435 (2009)). Three members of the Lateral Organ Boundary Domain gene family (LBD37, 38 and 39) were identified as nitrate-inducible genes and shown to be repressors of anthocyanin biosynthetic and nitrate assimilatory genes (Rubin G et al., Plant Cell, 21: 3567-3584 (2009)). Two kinase genes CIPK8 and CIPK23 were identified as nitrate-inducible genes. Mutations in CIPK8 result reduced gene induction at high but not low nitrate concentrations (Hu H C et al., Plant J, 57: 264-278 (2009)) while mutations in CIPK23 increase gene induction at low nitrate concentrations, indicating that these kinases selectively target different phases (i.e. high affinity versus low affinity phases) of the nitrate response. Interestingly, CIPK23 phosphorylates the nitrate transporter NRT1.1 (CHL1, (Tsay Y-F et al., Cell, 72: 705-713 (1993))), which has been shown to act as a nitrate regulator/sensor (Ho C H et al., Cell, 138: 1184-1194 (2009); Wang R et al., Plant Physiol., 151: 472-478 (2009); Krouk G et al., Curr Opin Plant Biol., 2010/01/23: 10.1016/j.pbi.2009.1012.1003 (2010)).
Even though several transcription factors have been identified, little is known about the cis-acting regulatory elements in nitrate-regulated promoters. One reason for the slow progress in identifying specific elements within nitrate enhancers is that no transient system has been reported in higher plants for rapid testing of nitrate-responsive promoters.